EP1919510B1 - Electrotransfert d'acide nucleique dans des cellules tissulaires - Google Patents

Electrotransfert d'acide nucleique dans des cellules tissulaires Download PDF

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EP1919510B1
EP1919510B1 EP06779983.3A EP06779983A EP1919510B1 EP 1919510 B1 EP1919510 B1 EP 1919510B1 EP 06779983 A EP06779983 A EP 06779983A EP 1919510 B1 EP1919510 B1 EP 1919510B1
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use according
volts
pulse
field strength
tissue
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EP1919510A2 (fr
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Luis Mir
Damijan Miklavcic
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Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Bioalliance Pharma SA
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Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Bioalliance Pharma SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0408—Use-related aspects
    • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
    • A61N1/0416—Anode and cathode
    • A61N1/042—Material of the electrode
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/325—Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention is related to the electrically mediated gene transfer of nucleic acids into tissue cells, in particular muscular or tumoral cells.
  • pulses usually are of lower voltage but much longer duration (in the range of tens of milliseconds) (Aihara and Miyazaki, 1998; Rols et al., 1998; Mir et al., 1999; Bettan et al ., 2000; Matsumoto et al., 2001). It is assumed that this type of pulses mediate DNA transfer into the cells by inducing two distinct effects that include cell permeabilization (like the short pulses) and DNA electrophoretic migration during the delivery of the electric field (Klenchin et al., 1991; Sukharev et al ., 1992; Neumann et al., 1996; Mir et al., 1999; Golzio et al., 2002).
  • Efficient electrotransfer into muscle cells has been described in WO-A-99/01158 using one or more (up to 100,000) unipolar electric impulsions of 1-800 volts/cm and in WO-A-98143702 using stimulation with an electric current of 5-200 volts/cm, wherein the electric current may be in the form of 2-30,000 square bipolar pulses.
  • Transfection of tumors and/or other tissues e.g. the liver can also be of interest for similar Preferred electric field strength (in V/cm) for the HV and/or the LV will change according to the tissues.
  • a first object of the invention is thus the use of a nucleic acid for the preparation of a human or veterinary medicament or drug intended to be transferred in vivo into tissue cells, wherein the medicament is brought into contact with tissue cells and the tissue is electrically stimulated as follows:
  • tissue denotes a tumoral or non tumoral tissue of an animal, for instance a human, or a non human Mammal such as a rodent (e.g. a mouse, a rabbit or a rat), a dog, a cat, or a primate.
  • a non tumoral tissue may be a muscle, especially skeletal muscle, or liver.
  • the tissue is a muscle.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 200 and 1400 volts/cm.
  • the tissue is a tumoral tissue.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 400 and 2000 volts/cm.
  • the medicament is intended to be brought into contact with the tissue cells before applying the single LV pulse and still more preferably, before the application of the HV pulse or pulses.
  • the time between injection of nucleic acid and electrical pulse, especially between injection and HV pulse or pulses, is not critical.
  • the medicament has been brought into contact with the tissue cells from few seconds to 10 minutes, e.g. from 30 s and 5 minutes. An interval of 5 to 10 minutes before the HV pulse or pulses is also acceptable.
  • the medicament may be brought into contact through direct intramuscular injection, through systemic administration (e.g. intravenous or intra-arterial route) or by topical or subcutaneous administration.
  • the single LV pulse has a field strength of between 50 and 140 volts/cm, especially of between 80 and 120 volts/cm, preferably of between 90 and 110 volts/cm, typically about 100 volts/cm.
  • the single LV pulse has a field strength of between 100 and 200 volts/cm, preferably of between 120 and 160 volts/cm, typically about 140 volts/cm.
  • the single LV pulse has a duration of between 300 and 800 ms, preferably of between 350 and 600 ms, typically about 400 ms.
  • the LV pulse may be of the same polarity than the HV pulse.
  • the LV pulse has a polarity opposed to that of the HV pulse.
  • the single LV pulse is a squared pulse. It can also be trapezoidal, or discontinuous.
  • the single LV pulse according to the invention at least improves the nucleic acid electrophoretic migration.
  • HV pulses There can be several HV pulses, i.e. from 2 to 10 HV pulses having the specifications disclosed therein. It is more convenient in this case to have identical HV pulses.
  • the HV pulse has a field strength of between 300 and 1300, preferably of between 400 and 1200 volts/cm, more preferably of between 500 and 900, still more preferably of between 600 and 800 volts/cm, typically about 700 volts/cm.
  • the HV pulse has a field strength of between 600 and 2000, preferably of between 800 and 1600 volts/cm, more preferably of between 900 and 1200, typically about 1000 volts/cm.
  • the HV pulse has a duration of between 10 and 1000 ⁇ s, preferably of between 50 and 200 ⁇ s, typically about 100 ⁇ s.
  • HV pulse it is preferably a squared pulse.
  • the HV and LV pulses may be separated by lag and this lag can advantageously be between 300 ms and 3000 s, preferably between 500 ms and 1000 s, typically about 1000 ms.
  • the HV pulse has a field strength of between 300 and 1000 volts/cm, preferably of between 400 and 800 volts/cm.
  • the nucleic acid is useful in gene therapy, either through expression of a molecule of interest or through modulation or blocking of a gene within the host that have a therapeutic effect.
  • the aims of transfection according to the invention are:
  • the nucleic acid comprises nucleic acid sequences able to express in vivo in the transfected tissue cells one or more therapeutically active molecule(s), preferably a protein or proteins of interest.
  • This active molecule may be therapeutically active by itself or indirectly e.g. through a metabolite of said molecule. It may acts in the tissue itself and/or outside the tissue in another location within the body, for example on a tumour located anywhere in the body if the expressed molecule is active against a tumour.
  • therapeutic molecules of interest one may refer to those listed in WO-A-99/01158 .
  • nucleic acid can be used, for example, plasmid DNA, linear DNA, antisense DNA and RNA.
  • the nucleic acid is a DNA expression vector of the type well known in the art.
  • an expression vector contains a promoter operably linked to a DNA sequence that codes for the protein of interest, followed by a termination signal such as a polyadenylation signal.
  • nucleic acids able to express in vivo different active molecules are used to prepare the medicament.
  • the nucleic acids are preferably chosen so as to be complementary and/or act in a synergistic way in treating a condition.
  • nucleic acid that is able to express in vivo at least two active molecules, that preferably are complementary and/or act in a synergistic way in treating a condition.
  • nucleotide sequences encoding the different molecules may be under the control of the same promoter or different promoters.
  • the nucleic acid expresses one or several (at least 2) active molecule(s) selected so that:
  • One embodiment is to transfect tissue, in particular muscle, cells with a construct comprising the Recombinant human Desintegrin Domain of ADAM-15 gene (RDD gene).
  • RDD gene Recombinant human Desintegrin Domain of ADAM-15 gene
  • This gene, its sequence and useful constructs e.g. expression vector pBi-RDD
  • the RDD gene and protein sequences are shown in SEQ ID No.1 and SEQ ID NO.2, respectively.
  • RDD may act as an anticancer agent, may reduce or suppress tumor growth, and/or acts as an antiangiogenic and/or antimetastatic agent.
  • a specific aspect of the invention is thus the use of a nucleic acid encoding the RDD protein or an efficient fragment thereof (efficient means the protein encoded by the fragment elicits the same or a similar therapeutic activity than the whole RDD polypeptide) for the preparation of a medicament intended to be transferred in vivo into tissue cells and to produce therein a RDD polypeptide or a fragment thereof that is therapeutically active, wherein the medicament is injected into a tissue and the tissue is electrically stimulated as follows:
  • the tissue is a muscle.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 200 and 1400 volts/cm.
  • the tissue is a tumoral tissue.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 400 and 2000 volts/cm.
  • This medicament is advantageously useful as an antiangiogenic and/or antimetastatic agent.
  • the nucleic acid encodes one or several immunogens (or immunogenic peptides, polypeptides or proteins, including glycoproteins) that are able to induce an immune response in the host.
  • the immune response is a protective immune response for the host.
  • the invention relates to producing an immunogenic composition or a vaccine or a therapeutic vaccine, that is directed against a microorganism, e.g. virus or bacteria, or against cancers.
  • the nucleic acid encodes one or several (at least 2) immunogens of HIV, HBV, Epstein-Barr virus, pseudorabies virus, syncitia forming virus.
  • the person skilled in the art has access to the nucleic acids encoding the most interesting molecules for the chosen application, for example to the most efficient immunogens or combinations of immunogens for a particular disease.
  • the immune response leads to the production of antibodies, especially polyclonal antibodies, and these antibodies are intended to be recovered from the produced serum and used in an usual manner.
  • the present invention also discloses a method of treatment of a Human or an animal, comprising injecting a nucleic acid into a tissue, and electrically stimulating the tissue as follows:
  • the tissue is a muscle.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 200 and 1400 volts/cm.
  • the tissue is a tumoral tissue.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 400 and 2000 volts/cm.
  • the nucleic acid is able once transferred in vivo into tissue cells to produce therein a therapeutically active molecule, that is intended to exert directly or indirectly a therapeutic action in the muscle cells and/or at another body location, or still in the tumor tissue cells.
  • the nucleic acid is injected before applying the single LV pulse and still more preferably, before the application of the HV pulse or pulses.
  • One aspect is thus such a method wherein the nucleic acid encodes the RDD gene or an efficient fragment thereof, as disclosed therein, and the method is intended to reduce or suppress tumor growth, and/or acts as an antiangiogenic and/or antimetastatic agent.
  • nucleic acid encodes an immunogen, as disclosed therein, and the method is intended to immunize a Human or an animal, or to produce antibodies to be recovered.
  • the invention also discloses an electroporation method, comprising placing electrodes near a tissue containing a nucleic acid interstitially, then electrically stimulating the tissue as follows:
  • the tissue is a muscle.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 200 and 1400 volts/cm.
  • the tissue is a tumoral tissue.
  • the tissue be electrically stimulated first with at least one pulse of a HV field strength of between 400 and 2000 volts/cm.
  • the nucleic acid is heterogeneous to the body and is of the type described supra. It is preferably a nucleic acid comprising nucleic acid sequences able to express in vivo in the transfected muscle cells or tumoral tissue one or more therapeutically active molecule(s), preferably a protein or proteins of interest.
  • the electrodes are placed at the contact of the skin, i.e. outside the body and this does not need any surgery act.
  • the electrodes are placed at the contact of the tissue, in particular the muscle or tumoral tissue, itself.
  • the electrodes may be carried by a device making both the injection of the nucleic acid and the electric stimulation.
  • the electrodes may also be separate from the injection device.
  • the electrodes are to be positioned near the injection site such that electrical current traveling through the electrodes passes through the injection site or region wherein the injected liquid has diffused upon injection.
  • the invention also relates to the use of a nucleic acid which is capable of expressing a molecule in the manufacture of a medicament or drug for use in a method of delivering said nucleic acid to tissue cells, especially tumoral or non tumoral tissue cells, e.g. muscle cells, wherein
  • Still another object of the invention is a method for the production of antibodies, especially polyclonal antibodies, comprising injecting an immunogen encoding nucleic acid into a tissue, especially a muscle, of a living animal and electrically stimulating the tissue as follows:
  • the animal may be a mice, a rat or a rabbit or any other animal especially rodent usually used for the production of antibodies.
  • Recovery of serum and antibodies, purifying and/or concentration of the antibodies may be done using the conventional methods known from the person killed in the art.
  • This method may be further defined with the various features defined above concerning especially the conditions of electrostimulation, of administration of the nucleic acid, the composition of the nucleic acid, the nature of the hosts...
  • the plasmid pXL 3031 (pCMV-Luc+) containing the cytomegalovirus promoter (nucleotides 229-890 of pcDNA3, Invitrogen) inserted upstream of the coding sequence of the modified cytosolic luc+ gene coding for the firefly luciferase (Soubrier et al., 1999) was used.
  • the plasmid DNA was prepared using usual procedures (Ausubel et al., 1994).
  • the pEGFP-N1 plasmid (BD Biosciences Clontech, Saint Quentin Yvelines, France) featuring the gene of the Green Fluorescent Protein (GFP) under the control of the CMV promoter and prepared in PBS (phosphate buffered saline, Gibco, Cergy-Pontoise, France) using the EndoFree Plasmid Giga Kit (QIAGEN, Courtabeuf, France) was also used.
  • PBS phosphate buffered saline, Gibco, Cergy-Pontoise, France
  • EndoFree Plasmid Giga Kit QIAGEN, Courtabeuf, France
  • mice were anesthetized by the intraperitoneal administration of the anesthetics Ketamine (100 mg/kg; Ketalar, Panpharma, France) and Xylazine (40 mg/kg; Rompun, Bayer, France). Prior to the experiments the legs were shaved using an electric shaver. At least 10 muscles (5 mice) were included in each experimental group for luciferase determinations. In the case of the GFP qualitative data, four muscles were used for each experimental condition.
  • plasmid DNA prepared in 30 ⁇ l of 0.9 % NaCl were injected.
  • the DNA solution was supplemented with 120 IU/ml heparin (Laboratoires Leo, Saint Quentin en Yvelines, France; one mg of the heparin (MW 10-12 kDa) corresponded to approximately 137 IU).
  • the DNA was injected into tibial cranial muscles using a Hamilton syringe with a 26-gauge needle.
  • 4 ⁇ g in 20 ⁇ l of PBS were injected in each treated tibialis, always in the absence of heparin.
  • HV and LV pulse combinations were generated by a device consisting of square wave electropulsator PS-15 (Jouan, St Herblain, France) for the HV and a microprocessor-driven switch/function generator built at the University of Ljubljana, Faculty of Electrical Engineering, Slovenia, for the LV.
  • PS-15 square wave electropulsator
  • the device allowed for precise control of every electrical parameter in HV+LV combinations of pulses (Satkauskas et al., 2002).
  • HV and LV pulse combinations were delivered soon (40 ⁇ 15 s) after intramuscular DNA injection. In all the experiments the lag between HV and LV was fixed to 1 s. For pulse delivery to the muscles stainless plate electrodes 4.4 mm apart were used. The 1-cm plates encompassed the whole leg of the mice. To ensure good contact between the tibial cranial muscle of exposed leg and the plates of the electrodes a conductive gel was used. Electric field values (in V/cm) are always expressed in terms of the ratio of the voltage applied (V) to the distance between the electrodes (cm).
  • the pulses combinations were delivered using a CLINIPORATORTM (IGEA, s. r. l., Carpi (MO), Italy) generator and 5 mm apart electrodes from the same company.
  • IGEA IGEA, s. r. l., Carpi (MO), Italy
  • mice were sacrificed 2 days after DNA electrotransfer.
  • the muscles (net weight approximately 60 mg) were took off and homogenized in 1 ml Cell Culture Lysis reagent solution (10 ml Cell Culture Lysis reagent (Promega Charbonippos, France), diluted with 40 ml distilled water and supplemented with 1 tablet of the Protease inhibitor cocktail from Boehringer Mannheim, Mannheim, Germany).
  • the luciferase activity was assessed on 10 ⁇ l of the supernatant, using a Walac Victor 2 luminometer, by integration of the light produced during 1 s, starting after the addition of 50 ⁇ l of Luciferase Assay Substrate (Promega) to the muscle lysate. The results were collected from the luminometer in relative light units (RLU). Calibration with purified firefly luciferase protein showed that 10 6 of RLU correspond to approximately 70 ng of expressed luciferase. The final results were expressed as pg of luciferase per muscle.
  • mice were sacrificed 3 days after the injection of the pEGFP-N1 plasmid and the transfected tissue was observed using a Leica MZ12 fluorescence stereomicroscope with a Leica GFP Plus filter set (Art. No. 10446143: excitation filter 480/40 nm, dichroic mirror 505 nm LP, barrier filter 510 nm LP) (Leica, Rueil-Malmaison, France). Pictures were taken using a digital cooled color camera (AxioCam HRc, Zeiss, Le Pecq, France), and the quantification of the GFP expression was made by software (AxioVision Light Edition Release 4.1.1.0) integration of the light detected by the camera.
  • the LV pulses giving the best level of gene expression according to previous data (Satkauskas et al., 2002) were used.
  • the LV component parameters were fixed for this experiment to four LVs of 80 V/cm and 100 ms duration, with a delay between the pulses of 1 second.
  • Fig. 1 As a consequence of the results shown in Fig. 1 , one single HV of 800 V/cm and 100 ⁇ s was always used to analyze the role of the LV component.
  • the LV pulse strength was fixed to 80 V/cm, duration to 100 ms and the delay between LVs to 1 s.
  • Luciferase expression markedly increased when LV number was increased from 1 to 4 ( Fig. 2 ). Consistently with previous data (Satkauskas et al., 2002), with four LVs the luciferase expression was 10 times higher than with one LV. No further significant increase was observed with a larger number (6 or 8) of LV pulses ( Fig. 2 ).
  • the expression vectors used in this experiment were prepared in accordance with Trochon-Joseph V. et al . 2004.
  • mice Legs of C57BL/6 mice were shaved using an electric shaver on the day before electrotransfer. Before the electrotransfer procedure, animals were anesthetized via the intraperitoneal injection of a mixture of ketamine (100mg/kg body weight) and xylazine (40mg/kg).
  • Plasmid mixture was injected using a Hamilton syringe. A conductive gel was applied to ensure good contact between the leg skin and the two stainless steel plate electrodes (space between the electrodes: 5 mm). Subsequently, one transcutaneous square-wave electric HV pulse of 700V/cm and 100 ⁇ s (1 Hz) was first applied to permeabilize membrane. After a 1000 ms pause and without moving electrodes, one transcutaneous square-wave electric LV pulse of 100V/cm and 400 ms was applied to allow DNA entry into cells by electrophoretic migration. Electrotransfer was performed with the electropulsator Cliniporator (IGEA, Italy). The same procedure was followed for each animal group and each leg. 10 mice were used in each group.
  • IGEA electropulsator Cliniporator
  • Figures 6 and 7 present the Luciferase expression after DNA electrotransfer into mice muscle tibialis using combinations of different HV pulses (200 to 1800 V/cm, 100 ⁇ s) followed by one LV pulse (80 V/cm; 400 ms) 1 s after the HV ( Figure 6 ) or immediately after the HV ( Figure 7 ). These experiments have been conducted as in example 1 for the luciferase protocol, on 6 mice for each group, using the CLINIPORATORTM to deliver the impulsions and the luciferase activity has been expressed in pg/mg of muscle.
  • B16 F10 melanoma cells were cultured in vitro using classical procedures and MEM culture medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 8% foetal calf serum.
  • Young (6-8 weeks) C57BI/6 female mice were inoculated subcutaneously in the left flank with 1x10 6 syngenic B16 cells (in 100 ⁇ l of MEM culture medium). The tumours were treated when they reached an average diameter of 6-7mm (7-8 days after inoculation).
  • the human RDD gene under the control of the murine urokinase secretion signal was inserted into the pVAX1 plasmid (Invitrogen, V260-20) containing the cytomegalovirus (CMV) promoter and the bovine growth hormone polyadenylation signal, to generate the pVAX-RDD plasmid.
  • the empty vector pVAX1 was used as a negative control. Plasmids were prepared in sterile 0.9% NaCl using the EndoFree NucleoSpin Plasmid Kit (Macherey Nagel)
  • Female Wistar rats were anesthetized by the intraperitoneal administration of the anesthetics Ketamine (40 mg/kg) and Xylazine (5.5 mg/kg).
  • Female New Zealand rabbits were first treated with subcutaneous injection of Calmivet (1ml/kg) and then anesthetized by intravenous injection of pentobarbital. Prior to the experiments, the legs were shaved using an electric shaver.
  • Penetrating needle electrodes were introduced into the Gracilis muscles for the rabbits and into the Gluteus muscles for the rats. 100 ⁇ g of each plasmid DNA prepared in 100 ⁇ l of 0.9% NaCl were injected in three times between the electrode lines. Immediately after intramuscular DNA injection, muscle electrotransfer with the CLINIPORATORTM was performed as previously described : one electric pulse of 700V/cm and 100 ⁇ s (1 Hz), a 1000 ms pause and, without moving electrodes, one electric pulse of 100V/cm and 400 ms. The procedure was performed for both legs of each animal, and twice per muscle for rabbits (i.e. 4 injections per animal) and once per muscle for rats (i.e. 2 injections per animal). Animals were immunized at 0, 6, and 12 week.
  • bound rat antibodies were detected with a goat F(ab')2 fragment rat IgG(H+L) peroxidase (ref. IM0825, Beckman Immunotech). After washing, as described above, wells were incubated with 200 ⁇ l of the substrate o-phenylenediamine dihydrochloride (Sigma Fast OPD peroxidase substrate tablet set) for 30 min. The reaction was stopped by adding 50 ⁇ l of 3N HCL, and a spectrophotometric reading was obtained at 490 nm.
  • substrate o-phenylenediamine dihydrochloride Sigma Fast OPD peroxidase substrate tablet set
  • the anti-RDD rabbit polyclonal serum used as a positive control was produces according to standard peptide immunization (production performed by Neosystem SA, France).
  • the selected peptide comprised amino acid residues 57 to 68 from the RDD sequence (SEQ ID NO.2) conjugated to Gluta-KLH (keyhole limpet hemocyanin, a carrier protein to enhance the immunogenicity of small peptides). 2 mg of this peptide were subcutaneously injected to rabbits at week 0, 2, 4 and 8. Blood were collected before the first injection, and then at week 6, 10, and 12, (sacrifice time). Serum collected at week 12 was used as the positive control.
  • anti-RDD IgG antibodies were produced at week 9 after immunization of rabbits and rats by muscle electrotransfers. This immunization by electrotransfer induced an anti-RDD antibody production as efficient than a classical immunization by peptide injection to rabbits (see control rabbit curve). Same results were obtained for rats at week 16.

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Claims (42)

  1. Utilisation d'un acide nucléique pour la préparation d'un médicament destiné à être transféré in vivo dans des cellules d'un tissu, où le médicament est mis en contact avec les cellules de tissu et le tissu est stimulé électriquement comme suit :
    - en premier avec au moins une impulsion d'une force de champ de haute tension entre 200 et 2000 volts/cm
    - en second avec une impulsion unique de force de champ de basse tension entre 50 et 200 volts/cm et d'une durée entre 300 et 2000 ms.
  2. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ entre 50 et 140 volts/cm.
  3. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ entre 80 et 120 volts/cm.
  4. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ entre 90 et 110 volts/cm.
  5. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ de 100 volts/cm.
  6. Utilisation selon l'une quelconque des revendications 1 à 4, où l'utilisation est faite d'une force de champ de haute tension entre 200 et 1400 volts/cm.
  7. Utilisation selon la revendication 6, où l'utilisation est faite d'une force de champ de haute tension entre 400 et 1200 volts/cm.
  8. Utilisation selon la revendication 6, où l'utilisation est faite d'une force de champ de haute tension entre 600 et 800 volts/cm.
  9. Utilisation selon la revendication 7, où l'utilisation est faite d'une force de champ de haute tension de 700 volts/cm.
  10. Utilisation selon l'une quelconque des revendications 1 à 9, où le tissu est un muscle.
  11. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ entre 100 et 200 volts/cm.
  12. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ entre 120 et 160 volts/cm.
  13. Utilisation selon la revendication 1, où l'impulsion unique de basse tension a une force de champ de 140 volts/cm.
  14. Utilisation selon l'une quelconque des revendications 1 et 11 à 13, où l'utilisation est faite d'une force de champ de haute tension entre 400 et 2000 volts/cm.
  15. Utilisation selon la revendication 14, où l'utilisation est faite d'une force de champ de haute tension entre 800 et 1600 volts/cm.
  16. Utilisation selon la revendication 14, où l'utilisation est faite d'une force de champ de haute tension entre 900 et 1200 volts/cm.
  17. Utilisation selon la revendication 14, où l'utilisation est faite d'une force de champ de haute tension de 1000 volts/cm.
  18. Utilisation selon l'une quelconque des revendications 1 et 11 à 17, où le tissu est un tissu tumoral.
  19. Utilisation selon l'une quelconque des revendications 1 à 18, où l'impulsion unique de basse tension a une durée entre 300 et 800 ms.
  20. Utilisation selon la revendication 19, où l'impulsion unique de basse tension a une durée entre 350 et 600 ms.
  21. Utilisation selon la revendication 19, où l'impulsion unique de basse tension a une durée de 400 ms.
  22. Utilisation selon l'une quelconque des revendications 1 à 21, où l'impulsion unique de basse tension a une polarité qui est opposée à celle de l'impulsion de haute tension.
  23. Utilisation selon l'une quelconque des revendications 1 à 22, où une impulsion de haute tension unique est utilisée.
  24. Utilisation selon l'une quelconque des revendications 1 à 23, où l'utilisation est faite d'une ou de plusieurs impulsions de champ de haute tension ayant une durée entre 10 et 1000 µs.
  25. Utilisation selon la revendication 24, où l'utilisation est faite d'une ou de plusieurs impulsions de champ de haute tension ayant une durée entre 50 et 200 µs.
  26. Utilisation selon la revendication 25, où l'utilisation est faite d'une ou de plusieurs impulsions de champ de haute tension ayant une durée de 100 µs.
  27. Utilisation selon l'une quelconque des revendications précédentes, où l'impulsion de haute tension et l'impulsion de basse tension sont séparées par un temps de latence.
  28. Utilisation selon la revendication 27, où le temps de latence est entre 300 ms et 3000 s.
  29. Utilisation selon la revendication 28, où le temps de latence est entre 500 ms et 1000 s.
  30. Utilisation selon la revendication 29, où le temps de latence est de 1000 ms.
  31. Utilisation selon l'une quelconque des revendications précédentes, où l'acide nucléique est capable de produire une molécule thérapeutiquement active ou plusieurs molécules thérapeutiquement actives dans les cellules de tissu.
  32. Utilisation selon l'une quelconque des revendications précédentes, où l'utilisation est faite de deux acides nucléiques ou plus capables de produire différentes molécules thérapeutiquement actives dans les cellules de tissu.
  33. Utilisation selon l'une quelconque des revendications précédentes, où l'acide nucléique code pour la protéine RDD ou un fragment efficace de celle-ci.
  34. Utilisation selon l'une quelconque des revendications précédentes, où le médicament est efficace dans la réduction ou la suppression d'une angiogenèse tumorale.
  35. Utilisation selon l'une quelconque des revendications précédentes, où le médicament réduit ou supprime une croissance tumorale.
  36. Utilisation selon l'une quelconque des revendications précédentes, où le médicament inhibe les métastases.
  37. Utilisation selon l'une quelconque des revendications précédentes, où le médicament agit contre un cancer.
  38. Utilisation selon l'une quelconque des revendications 1 à 10 et 19 à 32, où le tissu est un muscle et l'acide nucléique code pour un ou plusieurs immunogènes.
  39. Utilisation selon la revendication 38, où le(s) immunogène(s) est/sont un/des immunogène(s) du VIH.
  40. Utilisation selon l'une quelconque des revendications 1 à 10 et 19 à 37, où ledit tissu est un muscle.
  41. Utilisation d'un acide nucléique qui est capable d'exprimer une molécule, pour la fabrication d'un médicament pour une utilisation dans un procédé de délivrance dudit acide nucléique à des cellules d'un tissu, spécialement des cellules d'un tissu tumoral ou non tumoral, par exemple, des cellules musculaires, où
    a) ledit acide nucléique doit être injecté dans le tissu
    b) le tissu est stimulé électriquement comme suit :
    - en premier avec au moins une impulsion d'une force de champ de haute tension (HT) entre 200 et 2000 volts/cm
    - en second avec une impulsion unique de force de champ de basse tension (BT) entre 50 et 200 volts/cm et d'une durée entre 300 et 2000 ms.
  42. Procédé de production d'anticorps, spécialement d'anticorps polyclonaux, comprenant l'injection d'un acide nucléique codant pour un immunogène dans un tissu, spécialement un muscle, d'un animal vivant non humain et la stimulation électrique du tissu comme suit :
    - en premier avec au moins une impulsion d'une force de champ de haute tension (HT) entre 200 et 2000 volts/cm
    - en second avec une impulsion unique de force de champ de basse tension (BT) entre 50 et 140 volts/cm et d'une durée entre 300 et 2000 ms,
    l'acide nucléique étant transféré dans les cellules de tissu par résultat de cette stimulation électrique et l'expression dans ledit hôte d'un immunogène capable de déclencher une réponse immunitaire chez l'hôte, et la récupération des anticorps.
EP06779983.3A 2005-09-02 2006-09-01 Electrotransfert d'acide nucleique dans des cellules tissulaires Active EP1919510B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP06779983.3A EP1919510B1 (fr) 2005-09-02 2006-09-01 Electrotransfert d'acide nucleique dans des cellules tissulaires

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US71362305P 2005-09-02 2005-09-02
EP05291825A EP1759714A1 (fr) 2005-09-02 2005-09-02 Electrotransfert d'acides nucléiques dans des cellules tissulaires
PCT/IB2006/002401 WO2007026236A2 (fr) 2005-09-02 2006-09-01 Electrotransfert d'acide nucleique dans des cellules tissulaires
EP06779983.3A EP1919510B1 (fr) 2005-09-02 2006-09-01 Electrotransfert d'acide nucleique dans des cellules tissulaires

Publications (2)

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EP1919510A2 EP1919510A2 (fr) 2008-05-14
EP1919510B1 true EP1919510B1 (fr) 2014-03-05

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EP05291825A Withdrawn EP1759714A1 (fr) 2005-09-02 2005-09-02 Electrotransfert d'acides nucléiques dans des cellules tissulaires
EP06779983.3A Active EP1919510B1 (fr) 2005-09-02 2006-09-01 Electrotransfert d'acide nucleique dans des cellules tissulaires

Family Applications Before (1)

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EP05291825A Withdrawn EP1759714A1 (fr) 2005-09-02 2005-09-02 Electrotransfert d'acides nucléiques dans des cellules tissulaires

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US (1) US8227435B2 (fr)
EP (2) EP1759714A1 (fr)
JP (1) JP5165567B2 (fr)
CN (1) CN101252953A (fr)
AU (1) AU2006286306B2 (fr)
CA (1) CA2619783C (fr)
ES (1) ES2460949T3 (fr)
IL (1) IL189594A (fr)
WO (1) WO2007026236A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1970441A1 (fr) * 2007-03-06 2008-09-17 BioAlliance Pharma Plasmide contenant une séquence codant un domaine de disintégrine de métargidine (RDD)
US8855759B2 (en) * 2007-10-09 2014-10-07 The Hong Kong Polytechnic University Method of treating a rheumatic disorder using combination of transcutaneous electrical nerve stimulation and a ginsenoside
EP2353607A1 (fr) 2010-02-04 2011-08-10 BioAlliance Pharma Utilisation du domaine disintégrine d'une adamalysine pour le traitement du psoriasis
ES2753412T3 (es) 2013-10-28 2020-04-08 Invectys Electrotransferencia génica a las células de la piel

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PL337617A1 (en) * 1997-06-30 2000-08-28 Rhone Poulenc Rorer Sa Apparatus for performing optimised in vivo electrotransfer of vectorial nucleic acids into tissues
US6027488A (en) * 1998-06-03 2000-02-22 Genetronics, Inc. Flow-through electroporation system for ex vivo gene therapy
US6593130B1 (en) * 1999-04-16 2003-07-15 The Regents Of The University Of California Method and apparatus for ex vivo and in vivo cellular electroporation of gene protein or drug therapy
FR2827775B1 (fr) * 2001-07-26 2003-09-26 Bioalliance Pharma Utilisation dans une composition anti-angiogenique du domaine disintegrine d'adamalysine

Also Published As

Publication number Publication date
WO2007026236A8 (fr) 2013-04-25
IL189594A (en) 2012-08-30
CN101252953A (zh) 2008-08-27
US8227435B2 (en) 2012-07-24
WO2007026236A3 (fr) 2007-05-03
WO2007026236A2 (fr) 2007-03-08
AU2006286306A1 (en) 2007-03-08
ES2460949T3 (es) 2014-05-16
CA2619783C (fr) 2016-03-22
EP1919510A2 (fr) 2008-05-14
AU2006286306B2 (en) 2012-04-19
US20080027018A1 (en) 2008-01-31
JP5165567B2 (ja) 2013-03-21
CA2619783A1 (fr) 2007-03-08
EP1759714A1 (fr) 2007-03-07
IL189594A0 (en) 2008-08-07
JP2009507786A (ja) 2009-02-26

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